Chemokines are small secreted pro-inflammatory proteins, which mediate directional migration of leukocytes from the blood to the site of injury. Depending on the position of the conserved cysteines characterizing this family of proteins, the chemokine family can be divided structurally in C, C-C, C-X-C and C-X3-C chemokines, to which corresponds a series of membrane receptors (Baggiolini M et al., 1997; Fernandez E J and Lolis E, 2002). Usually chemokines are produced at the site of injury and cause leukocyte migration and activation, playing a fundamental role in inflammatory, immune, homeostatic and angiogenic processes. These molecules, therefore, offer the possibility for therapeutic intervention in diseases associated to such processes, in particular by inhibiting specific chemokines and their receptors at the scope to preventing the excessive recruitment and activation of leukocytes (Baggiolini M, 2001; Loetscher P and Clark-Lewis I, 2001; Godessart N and Kunkel S L, 2001).
Monocyte chemoattractant protein 1 (from now on, MCP-1) is a member of the CC chemokine family also known under various names such as CCL2, Small Inducible Cytokine A2 (SCYA2), Monocyte Chemotactic And Activating Factor (MCAF), Monocyte Secretory Protein Je, Monocyte Chemotactic Factor, and HC11. This chemokine is capable of promoting the recruitment of monocytes and basophils in response to injury and infection signals in various inflammatory diseases, different types of tumors, cardiac allograft, AIDS, and tuberculosis (Gu L et al., 1999).
Structurally and functionally homologous proteins have been identified and called MCP-2 (CCL7), MCP-3 (CCL8), MCP-4 (CCL13), and Eotaxin (CCL11). This subfamily of C-C chemokines is significantly distinct from other C-C chemokines, such as RANTES or MIP-1alpha/beta, and probably coevolved from a common progenitor sequence. They have a similar receptor usage, binding in particular CCR2 (but also for CCR1, CCR3, and CCR5). Therefore, many of the immunological and inflammatory agonistic or antagonistic activities of these C-C chemokines are common (Hughes A L and Yeager M, 1999; Berhkout T A et al., 1997; Luster A D and Rothenberg M E, 1997, Proost P et al., 1996).
The physiological activities associated with MCP-1 have been extensively studied by means of transgenic animals and other animal models, which demonstrate that MCP-1 controls recruitment of monocytes and of other cell types (astrocytes, for example) in many infectious, inflammatory and autoimmune diseases, as well as the expression of cytokines related to T helper responses. Other diseases that appear induced by MCP-1 are vascular disorders (restenosis after coronary intervention, arteriosclerosis, atherosclerosis, ischemia, stroke) and cancer-related angiogenesis (Ikeda Y et al., 2002; Egashira K et al., 2002; Gu L et al., 2000; Salcedo R et al., 2000; Gosling J et al, 1999; Lu B et al, 1998; Rutledge B J et al., 1995).
Since MCP-1 targeting is considered as a possible therapeutic approach for several diseases, different types of MCP-1 antagonists have been described in the literature, obtaining more or less important inhibitory effect on MCP-1-induced pathological activities (Dawson J, 2003). Examples of MCP-1 antagonists are an N-terminal deletion mutant of MCP-1, natural or synthetic, missing the N-terminal amino acids 2 to 10 (Egashira K et al., 2000; Zhang Y and Rollins B J, 1995; McQuibban G A et al., 2002), anti-MCP-1 monoclonal antibodies (Ajuebor M N et al., 1998; Eghtesad M et al., 2001), RNA aptamers (Rhodes A et al., 2001), peptides designed on sequences internal to MCP-1 (Reckless J and Grainger D J, 1999), MCP-1 antagonists peptide mimics (Kaji M et al., 2001), antisense oligonucleotides (WO 94/09128), small molecules (Mirzadegan T et al., 2000), polymer-modified MCP-1 (WO 02/04015), or viral decoy receptors (Alexander J M et al., 2002; Beck C G et al., 2001).
Structurally, MCP proteins present a N-terminal loop and three β-sheets overlaid by a α-helix at the C-terminal end (Handel T M et al., 1996; Lubkowski J, et al., 1997; Blaszczyk J et al., 2000). The literature provides many examples of structure-activity studies (Gong J H and Clark-Lewis 1, 1995; Zhang et al., 1996; Beall C J et al., 1996; Steitz S A et al., 1998; Gu L et al., 1999; Hemmerich S et al., 1999; Seet B T et al., 2001) in which MCP-1 mutants have reduced activity and/or affinity for the receptor or other binding proteins have been obtained by expressing N-terminal truncations (as in many other chemokines), or single mutations at residues 3, 8, 10, 13, 15, 18, 19, 24, 28, 30, 37, 38, and 39 (following the numbering of mature human MCP-1). Similar results have been obtained for Eotaxin (Mayer M R and Stone M J, 2001).
Chemokines interact with proteoglycans (PGs) and glycosaminoglycans (GAGs) a feature common to many cell-signaling soluble molecules (interleukins, growth factors). Proteoglycans are negatively charged proteins that are post-translationally modified by the addition of glycosaminoglycan side chains at serine residues. Clusters of basic residues (mainly Arginines and Lysines) allow proteins to interact with GAGs, which commonly are characterized by the disaccharide repeats such as heparin, chondroitin sulfate, heparan sulfate, dermatan sulfate, and hyaluronic acid). PGs and GAGs can be present on membrane surfaces as well as soluble molecules, probably at the scope to protect this molecule from proteolysis in the extracellular environment. It has been also proposed that GAGs may help the correct presentation of cell signaling molecules to their specific receptor and, eventually, also the modulation of target cell activation. In the case of chemokines, the concentration into immobilized gradients at the site of inflammation and, consequently, the interaction with cell receptors and their activation state seem to be modulated by the specific GAG. The interaction with GAGs and the formation of these gradients have been clearly demonstrated for many chemokines, including MCP-1, measuring the relative affinity. Therefore, it has been suggested that the modulation of such interactions may represent a therapeutic approach in inflammatory disease (Hoogewerf A J et al., 1997; Kuschert G et al., 1999; Ali S et al., 2001; Patel D et al., 2001; WO 02/28419; WO 99/50246).
However, the structural requirements and functional effects of GAG/MCP-1 interactions have been poorly studied. It is known that GAGs can modulate the activity and production of MCP-1 secreted from endothelial cells (Douglas M S et al., 1997). It has been also reported that substitution of Lysine 58 and Histidine 66 with Alanines in the C-terminal of MCP-1, prevents GAG binding without affecting receptor binding, Ca2+ influx, or chemotactic activity (Chakravarty L et al, 1998), but there is no disclosure in the prior art of which may be other GAG binding sites of MCP-1, and which in vivo effects can be consequent to their elimination. Even though extensive studies have been performed on some chemokines, it is not possible to anticipate, on the basis of the sequence homology, which residues have to be modified with non-conservative substitutions to impair GAG binding, and which effects can be obtained, since there is a significant structural diversity of GAG binding domains in chemokines (Lortat-Jacob H et al., 2002).